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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4179-4186
Different Effect of Various Mutant MITF Encoded by mi,
Mior, or Miwh Allele on
Phenotype of Murine Mast Cells
By
Dae-Ki Kim,
Eiichi Morii,
Hideki Ogihara,
Young-Mi Lee,
Tomoko Jippo,
Shiro Adachi,
Kazutaka Maeyama,
Hyung-Min Kim, and
Yukihiko Kitamura
From the Department of Pathology, Osaka University Medical School,
Osaka, Japan; the Department of Pharmacology, Ehime University Medical
School, Ehime, Japan; and the Department of Oriental Pharmacy, College
of Pharmacy, Wonkwang University, Chonbuk, Republic of Korea.
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ABSTRACT |
The mi locus encodes a member of the
basic-helix-loop-helix-leucine zipper protein family of transcription
factors (hereafter called MITF). Mutant alleles of mi,
Mior, and Miwh are deletion or
point mutation of the basic domain by which MITF binds DNA. The basic
domain also has nuclear localization potential. In the present study,
we compared the mast cell abnormalities of
Mior/Mior and
Miwh/Miwh mice with those of
mi/mi mice, of which many have been described by us. The number
of mast cells in the skin of Mior/Mior
suckling mice was remarkably decreased from that observed in mi/mi suckling mice, but the number was normal in the skin of Miwh/Miwh suckling mice. The decrease
in skin mast cells was more severe in the mi/mi embryos than in
mi/mi suckling mice, but the magnitude of the decrease was
comparable between Mior/Mior embryos
and Mior/Mior suckling mice. The poor
mRNA expression of granzyme B and tryptophan hydroxylase genes was
observed in all cultured mast cells (CMCs) derived from the spleens of
Miwh/Miwh,
Mior/Mior, and mi/mi mice.
However, the poor expression of mouse mast cell protease-4 (MMCP-4),
MMCP-5, and MMCP-6 was observed only in
Mior/Mior and mi/mi CMCs. MITF
encoded by Miwh mutant allele
(Miwh-MITF) showed deficient but demonstratable DNA
binding, but mi-MITF and Mior-MITF did not
show any DNA binding ability. Although Miwh-MITF
and Mior-MITF showed normal nuclear localization
potential, the potential was significantly impaired in mi-MITF.
The rank order of mast cell abnormality (mi/mi > Mior/Mior > Miwh/Miwh) appears to be related to the
functional abnormality of MITF encoded by each mutant gene.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE mi LOCUS ENCODES a member of
the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of
transcription factors (hereafter called mi-transcription factor
[MITF]).1,2 Spontaneous, chemical, radiation, and
insertional mutageneses provided abundant mutant alleles at the
mi locus.3,4 More than 10 mutants are known to
exist at the mi locus, and half of them have been characterized
at the molecular level.5,6 The allele that has been studied
most intensively is the mi mutant allele. The mi/mi
mice show depletion of pigment in both the hair and eyes,
microphthalmia, and osteopetrosis.3,4 In addition, the
number of mast cells decreases and their phenotype is abnormal in
mi/mi mice.7-13 Although most mast cells in the
skin of normal (+/+) mice are stained with berberine sulfate that binds
heparin proteoglycan, few mast cells are berberine sulfate positive in the skin of mi/mi mice.11,14-17 Cultured mast cells
(CMCs) derived from the spleen of mi/mi mice are deficient in
the expression of various genes, such as the mouse mast cell protease-6
(MMCP-6),18 MMCP-5,19
c-kit,20 p75 nerve growth factor
receptor,21 granzyme B (Gr B),22 tryptophan
hydroxylase (TPH),22 and integrin  4 subunit
genes.23 MITF transactivates the genes whose expression is
deficient in mi/mi CMCs.18-23
MITF encoded by the mi mutant allele (mi-MITF) deletes
1 of 4 consecutive arginines in the basic domain.1,5,6 The
mi-MITF is defective in DNA binding ability and nuclear
localization potential.24,25 Because of these two
abnormalities, the mi-MITF does not transactivate target
genes.18-23,25 Mior and
Miwh are other mutant alleles at the mi
locus.3,4 As in the case of mi-MITF, MITF encoded
by the Mior or Miwh mutant
allele (or-MITF or wh-MITF, respectively) also
possesses a mutation at the basic domain.5,6 Arginine
changes to lysine at codon 216 in or-MITF, whereas isoleucine
changes to aspargine at codon 212 in wh-MITF. In contrast to
the detailed molecular study of the mi-MITF, few studies have
been performed regarding or-MITF or wh-MITF. The
depletion of pigment and microphthalmia of
Mior/Mior mice are not distinguishable
from those of mi/mi mice, whereas the degree of osteopetrosis
is milder in Mior/Mior mice than in
mi/mi mice.3,4,26 The depletion of pigment of
Miwh/Miwh mice is not distinguishable
from that of Mior/Mior and
mi/mi mice, whereas the degree of microphthalmia is milder in
Miwh/Miwh mice than in
Mior/Mior mice.3,4
Moreover, osteopetrosis is not detectable in
Miwh/Miwh mice.4
Abnormalities of mast cells have not been studied in Mior/Mior and
Miwh/Miwh mice.
In the present study, we compared the number of mast cells in the skin
of Mior/Mior and
Miwh/Miwh mice with that in the skin of
mi/mi and +/+ mice. We also compared the phenotype of skin mast
cells and that of CMCs derived from the spleen among
Mior/Mior,
Miwh/Miwh, mi/mi, and +/+ mice. The
rank order of mast cell abnormalities was mi/mi > Mior/Mior > Miwh/Miwh; this rank order was the same
as that of the severity observed in microphthalmia and osteopetrosis.
The rank order appeared to be explained by the molecular abnormality of MITF.
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MATERIALS AND METHODS |
Mice.
A diagram of the mi locus with its theoretical functional
domains and the location of the mutations investigated in this study is
shown in Fig 1. The original stock of
C57BL/6-mi/+ (mi/+) and C57BL/6-Miwh/+ (Miwh/+) mice
was purchased from the Jackson Laboratory (Bar Harbor, ME), and the
original stock of C57BL/6-Mior/+
(Mior/+) mice was kindly given by Dr L. Lamoreux
(Texas A&M University, College Station, TX). They were maintained in
our laboratory by consecutive backcrosses to our own inbred C57BL/6
colony (>15 generations at the time of the present experiment in
mi/+ and Miwh/+ mice and >10 generations
in Mior/+ mice). Female and male heterozygous mice
were crossed together, and the resulting homozygous mice were selected
by their white coat color.3,4 C57BL/6-+/+ (+/+) mice raised
in our laboratory were used as a control.
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| Fig 1.
A diagram of MITF with its theoretical functional domains
and the location of the mutations investigated in this study. The
mutation point at or-MITF or wh-MITF was boxed. The
amino acids are numbered from the initiation codon. The mi-MITF
deletes one of three arginines (amino acid 215 to 217). It is unclear
which one has been deleted.
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Cells.
Pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM)
was prepared according to the method described by Nakahata et
al.27 PWM-SCM may contain interleukin-3 (IL-3), IL-4, IL-6, IL-9, IL-12, IL-13, granulocyte-macrophage colony-stimulating factor,
macrophage colony-stimulating factor, tumor necrosis factor ,
transforming growth factor  , interferon  , and insulin growth factor 1.28 Mice of mi/mi,
Mior/Mior,
Miwh/Miwh, and control +/+ were used at
2 to 3 weeks of age to obtain CMCs. Mice were killed by decapitation
after ether anesthesia and spleens were removed. Spleen cells were
cultured in -minimal essential medium (-MEM; ICN Biomedicals,
Costa Mesa, CA) supplemented with 10% PWM-SCM and 10% fetal calf
serum (FCS; Nippon Bio-supp Center, Tokyo, Japan). Half of the medium
was replaced every 5 days. The number of cells derived from the spleen
of each mutant mice was 1 × 107 within 4 weeks.
Greater than 95% of cells contained alcian blue-positive granules and
were considered to be CMCs 4 weeks after initiation of the culture.
Three independent cultures were examined for each mutant. The 293T
cells were provided by Dr D. Baltimore (Rockefeller University, New
York, NY) and were maintained in Dulbecco's modification of Eagle's
medium (DMEM; Flow Laboratories, Irvine, UK) supplemented with 10% FCS.
Staining and counting of mast cells.
Pregnant mice on day 18 postcoitum (pc) were killed by decapitation
after ether anesthesia, and embryos were removed, fixed in Carnoy's
solution, and embedded in paraffin. Twenty days after birth, mice were
also killed by decapitation after ether anesthesia. Pieces
of dorsal skin were removed, smoothed onto a piece of the filter paper
to keep them flat, fixed in Carnoy's solution, and embedded in
paraffin. Sections of embryos and skin pieces were stained with alcian
blue or with berberine sulfate. The staining method with alcian blue or
berberine sulfate has been described previously.9,14-16
Mast cells between the epithelium and panniculus carnosus were counted
under the microscope, and the number was expressed as mast cells per
centimeter of skin. Berberine sulfate-positive mast cells were counted
under the fluorescent microscope, and the proportion of berberine
sulfate-positive cells to alcian blue-positive cells was calculated.
In situ hybridization.
Skin pieces were removed from the back of 20-day-old mice, fixed in 4%
paraformaldehyde in 0.1 mol/L phosphate buffer (PB; pH 7.4), and
embedded in paraffin. The technique of in situ hybridization has been
described in detail.29 To obtain the MMCP-4,30
MMCP-6,31 and MC-CPA probes,32 single-stranded
cDNA was generated from total RNA extracted from CMCs from +/+ mice
using the lithium chloride-urea method.33 The specific cDNA
of proteases was then amplified with specific primers for each protease
by polymerase chain reaction (PCR).13 The cDNAs were
subcloned into the EcoRV site of Bluescript KS ( )
plasmid (pBS; Stratagene, La Jolla, CA) that contains T3 and T7
promoters to generate probes. The number of various protease
mRNA-positive cells and that of alcian blue-positive cells were counted
in the serial sections, and the proportion of various protease
mRNA-positive cells to alcian blue-positive cells was calculated.
Northern blot analysis.
Each RNA sample was prepared from 1 × 107 of CMCs
using the lithium chloride-urea method.33 Northern blot
analysis was performed using MMCP-4,30
MMCP-5,34 MMCP-6,31
c-kit,35 TPH,22 Gr B,22 and
GAPDH36 cDNAs labeled with -[32P]-dCTP
(DuPont/NEN Research Products, Boston, MA; 10 mCi/mL) by random
oligonucleotide priming. After hybridization at 42°C, blots were
washed to a final stringency of 0.2× SSC (1× SSC is 150 mmol/L NaCl and 15 mmol/L trisodium citrate, pH 7.4) and subjected to autoradiography.
Concentration of serotonin.
The concentration of serotonin was measured using high-performance
liquid chromatography (HPLC) with electrochemical
detection.37 Briefly, CMCs were collected, washed with
phosphate-buffered saline (PBS), counted, and sonicated for 20 seconds
in a sonicator (Tomy, Tokyo, Japan) in 1 mL of ice-cold 3% perchloric
acid containing 5 mmol/L EDTA and 1 mmol/L sodium metabissulfate. The
homogenate was centrifuged at 10,000g for 15 minutes at 4°C
and the supernatant was applied directly to the HPLC column. The
concentration of serotonin per 1.0 × 106 cells was calculated.
Flow cytometry.
CMCs were harvested and washed once with cold PBS containing 0.5%
bovine serum albumin (BSA) and 0.1% sodium azide. The
cells were incubated with rat anti-c-kit monoclonal antibody
(MoAb) ACK238,39 (kindly given by Dr S.I. Nishikawa, Kyoto
University, Kyoto, Japan) at 4°C for 30 minutes, rinsed, developed
with fluorescein isothiocyanate (FITC)-conjugated rabbit antirat IgG,
and then analyzed on a FACScan (Becton Dickinson, Los Angeles, CA).
Immunohistochemistry.
Freshly dissected tissue samples were covered with tissue-Tek OCT
compound (an embedding medium to freeze tissues; Miles Inc, Elkhart,
IN) and frozen in liquid nitrogen. Frozen sections (4 µm thick) were
prepared with a cryostat. The sections were fixed with ice-cold
methanol-acetone (1:1), washed in PBS (pH 7.5), and incubated with ACK2
MoAb at the concentration of 10 µg/mL in PBS overnight at 4°C.
The sections were than washed five times with PBS and incubated with
FITC-conjugated rabbit antirat IgG antibody (DAKO A/S, Glostrup,
Denmark) for 40 minutes at 4°C, followed by extensive washing with
PBS. The specimens were examined using a confocal laser scanning
microscope (LSM-GM200; Olympus, Tokyo, Japan). After examination with
the confocal laser scanning microscope, the specimens were washed and
stained with alcian blue and nuclear fast red.
Evaluation of cellular response.
Cellular response was quantified by an
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT;
Sigma Chemical, St Louis, MO) rapid colorimetric assay as described
previously.40,41 Briefly, triplicate aliquots of cells (5 × 104) suspended in 100 µL of Cosmedium-001 (Cosmo
Bio Co, Tokyo, Japan) were cultured in 96-well microtiter plates for 48 hours at 37°C with various concentrations of recombinant mouse stem
cell factor (rmSCF). rmSCF was a generous gift of Kirin
Brewery Co Ltd (Tokyo, Japan). MTT (10 µL of 5 mg/mL solution of MTT
in PBS) was added to all wells for the final 4 hours of the culture.
Acid isopropanol (100 µL of 0.04 N HCl in isopropanol) was added to
all of the wells and mixed to dissolve the dark blue crystals. The
optical density was then measured on a Multiskan MCC/340MKII
(Labsystems, Helsinki, Finland) with a test wavelength of 540 nm and a
reference wavelength of 620 nm.
Construction of expression plasmids and immunocytochemistry.
The pBS containing the whole coding region of +-MITF,
mi-MITF, or-MITF, or wh-MITF was constructed in
our laboratory18 (hereafter called pBS-+-MITF,
pBS-mi-MITF, pBS-or-MITF, and pBS-wh-MITF, respectively). pEF-BOS expression vector was kindly provided by Dr S. Nagata42 (Osaka University, Osaka, Japan). The Sma
I-HincII fragment of pBS-+-MITF, pBS-mi-MITF,
pBS-or-MITF, or pBS-wh-MITF was introduced into the
blunted Xba I site of pEF-BOS. The expression plasmid was
transfected into 293T cells, and the overexpressed MITF protein was
detected by anti-MITF polyclonal antibody as described
previously.25 Briefly, the cells were fixed with 100% methanol, permealized by treatment with 0.2% Triton X-100
in PBS, and incubated with the rabbit anti-MITF polyclonal antibody.
The specimens were washed with PBS and then incubated with the
biotin-conjugated goat antirabbit IgG antibody (DAKO A/S).
Immuno-reacted cells were visualized with
streptavidin-biotin-peroxidase and 0.05% diaminobenzidine-0.02%
hydroperoxide solution (DAKO A/S) according to the manufacturer's instructions.
Electrophoretic gel mobility shift assay (EGMSA).
The production of the fusion protein containing
glutathione-S-transferase (GST) and MITF was described
previously.24 Oligonucleotides were labeled with
-[32P]-dCTP by filling 5'-overhangs and were
used as probes of EGMSA. DNA-binding assays were performed in a 20 µL
reaction mixture containing 10 mmol/L Tris-HCl (pH 8.0), 1 mmol/L
ethylenediaminetetraacetic acid (EDTA), 75 mmol/L KCl, 1 mmol/L
dithiothreitol, 4% Ficoll type 400, 50 ng of poly (dI-dC), 25 ng of
labeled DNA probe, and 3.5 µg of GST-MITF fusion protein. After
incubation at room temperature for 15 minutes, the reaction mixture was
subjected to electrophoresis at 14 V/cm at 4°C on a 5%
polyacrylamide gel in 0.25× TBE buffer (1× TBE is 90 mmol/L
Tris-HCl, 64.6 mmol/L boric acid, and 2.5 mmol/L EDTA, pH 8.3). The
polyacrylamide gels were dried on Whatman 3MM chromatography paper
(Whatman International Ltd, Maidstone, UK) and subjected
to autoradiography.
Dissociation assay.
Dissociation assay was performed according to Bousset et
al.43 Reaction mixtures for the DNA-+-MITF and
DNA-wh-MITF complexes were prepared as described above, and one
sixth of each reaction mixture was electrophoresed. A 500-fold molar
excess of nonlabeled oligonucleotide containing the CACATG motif was
then added, and the reactions were sampled after further incubation
times of 30, 60, 90, 120, and 150 seconds. The regions of the
autoradiogram containing DNA-+-MITF or DNA-wh-MITF complex were
scanned with a densitometer (Molecular Dynamics, Sunnyvale, CA), and
the values were plotted against the incubation period.
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RESULTS |
The number of mast cells was examined in the skin of embryos and
suckling mice of +/+, mi/mi,
Mior/Mior, and
Miwh/Miwh genotype. Histological
sections of day-18 pc embryos and the skin pieces of 20-day-old mice
were stained with alcian blue. In both day-18 pc
Mior/Mior embryos and 20-day-old
Mior/Mior mice, the number of mast
cells decreased to one third that of +/+ mice
(Table 1). The number of mast cells in
day-18 pc Mior/Mior embryos was greater
than that of day-18 pc mi/mi embryos, whereas the number of
mast cells in 20-day-old Mior/Mior mice
was comparable to that of 20-day-old mi/mi mice. The number of
mast cells was normal in day-18 pc
Miwh/Miwh embryos and 20-day-old
Miwh/Miwh mice (Table 1).
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Table 1.
Number of Mast Cells in the Skin of Day-18 pc Embryos
and 20-Day-Old Mice of +/+, mi/mi,
Mior/Mior, and
Miwh/Miwh Genotype
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Mature skin mast cells of +/+ mice contain heparin. Because mast cells
stained with berberine sulfate are considered to contain heparin,
proportion of berberne sulfate positive mast cells was used as an index
of maturation. In day-18 pc +/+ embryos, approximately half of the skin
mast cells were berberine sulfate positive, whereas only a few percent
of mast cells were berberine sulfate positive in day-18 pc
mi/mi and Mior/Mior embryos
(Table 2). The proportion of berberine sulfate-positive mast cells in day-18 pc Miwh/Miwh
embryos was comparable to that of day-18 pc +/+ embryos. In 20-day-old +/+ mice, most of skin mast cells were berberine sulfate positive, but
the proportions of berberine sulfate positive mast cells remained 3%
and 36% in 20-day-old mi/mi and
Mior/Mior mice, respectively. The
proportions of berberine sulfate-positive mast cells in day-18 pc
Miwh/Miwh embryos and 20-day-old
Miwh/Miwh mice was comparable to those
of +/+ mice of the same age (Table 2).
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Table 2.
Proportion of Berberine Sulfate-Positive to Alcian
Blue-Positive Cells in the Skin of Day-18 pc Embryos and 20-Day-Old
Mice of +/+, mi/mi, Mior/Mior, and
Miwh/Miwh Genotype
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The expression of mast cell-specific protease genes was analyzed by in
situ hybridization in mast cells of the skin. In mi/mi and
Mior/Mior mice, the proportion of
MMCP-4 or MMCP-6 mRNA+ mast cells decreased remarkably, but
that of MMCP-5 or MC-CPA mRNA+ mast cells did not
(Table 3). The skin mast cells of
Miwh/Miwh mice normally expressed all
protease genes examined.
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Table 3.
Proportion of MMCP-4, MMCP-5, MMCP-6, and MC-CPA
mRNA+ Cells to Alcian Blue-Positive Cells in the Skin of
+/+, mi/mi, Mior/Mior, and
Miwh/Miwh 20-Day-Old Mice
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The expression of genes that had been demonstrated to be affected by
MITF was examined in CMCs derived from the spleen of +/+,
mi/mi, Mior/Mior, and
Miwh/Miwh mice. As shown
previously,18-22 the expression of all examined genes was
hardly detectable in mi/mi CMCs. In
Mior/Mior CMCs, the expression of
MMCP-4, MMCP-5, MMCP-6, and TPH genes was hardly detectable, whereas
the expression of Gr B and c-kit genes of moderate degree was
detectable (Fig 2). In
Miwh/Miwh CMCs, expression levels of
MMCP-4, MMCP-5, MMCP-6, and c-kit genes were comparable to
those of +/+ CMCs, whereas the expression levels of Gr B and TPH genes
decreased significantly (Fig 2). The expression levels of Gr B and TPH
genes in Miwh/Miwh CMCs were comparable
to those of Mior/Mior CMCs.
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| Fig 2.
Expression of various genes in CMCs derived from +/+,
mi/mi, Mior/Mior, and
Miwh/Miwh mice. (A) The blot was
hybridized with 32P-labeled cDNA probe of MMCP-4, MMCP-5,
MMCP-6, c-kit, Gr B, TPH, or GAPDH. (B) Quantification of
amount of each mRNA using densitometry. Three independent experiments
were performed, and comparable results were obtained. A representative
experiment is shown.
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The expression of MMCP-5 gene was detectable in skin mast cells but not
in CMCs of mi/mi and Mior/Mior
mice (Table 3 and Fig 2). We previously reported that the addition of
SCF significantly increased the amount of MMCP-5 mRNA in mi/mi CMCs and speculated that SCF synthesized by fibroblasts may induce the
MMCP-5 expression in mi/mi skin mast cells.19 The
different pattern of MMCP-5 expression between CMCs and skin mast cells of mi/mi and Mior/Mior mice may
be attributable to the different concentration of SCF surrounding mast cells.
The serotonin content of +/+, mi/mi,
Mior/Mior, and
Miwh/Miwh CMCs was compared, because
TPH is the rate-limiting enzyme of the serotonin synthesis.44 The serotonin content was significantly
smaller in mi/mi, Mior/Mior,
and Miwh/Miwh CMCs than in +/+ CMCs
(Table
4).
The expression of c-kit protein in CMCs was examined by flow
cytometry. The expression level of c-kit protein decreased
significantly in Mior/Mior and
mi/mi CMCs, and the magnitude of decrease was more remarkable in mi/mi CMCs than in Mior/Mior
CMCs (Fig 3A). The expression level of
c-kit protein in Miwh/Miwh CMCs
was comparable to that of +/+ CMCs (Fig 3A). Next, the expression level
of c-kit protein was compared in the skin mast cells of day-18
pc embryos and 20-day-old mice by using the confocal laser scanning
microscope. At least 20 fields were examined in three tissue samples.
In skin mast cells of day-18 pc embryos, the expression level of
c-kit protein decreased significantly in
Mior/Mior and mi/mi genotypes;
the magnitude of decrease was more remarkable in mi/mi embryos
than in Mior/Mior embryos (Fig 3B). The
skin mast cells of day-18 pc Miwh/Miwh
embryos expressed a level of c-kit protein comparable to that of +/+ embryos. In skin mast cells of 20-day-old mice, the expression level of c-kit protein decreased in the
Mior/Mior and mi/mi genotype
(Fig 3B). The expression level of c-kit protein of
Miwh/Miwh mice was comparable to that
of +/+ mice. The cellular response of +/+, mi/mi,
Mior/Mior, and
Miwh/Miwh CMCs to rmSCF was then
compared using the MTT method (Fig 4). The
+/+ CMCs and Miwh/Miwh CMCs responded
to rmSCF in a dose-dependent manner, but
Mior/Mior CMCs and mi/mi CMCs
did not (Fig 4).
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| Fig 3.
Expression of c-kit protein in CMCs and skin mast
cells. (A) Flow cytometry of the surface expression of c-kit
protein in CMCs derived from +/+, mi/mi,
Mior/Mior, and
Miwh/Miwh mice. Cells were incubated
with either ACK2 MoAb or negative control antibody. (B) The
representative field showing the content of c-kit protein in
skin mast cells of day-18 pc embryos or 20-day-old mice. Specimens were
observed by the confocal laser scanning microscope after staining
c-kit protein with ACK2 MoAb. At least 20 fields were examined
in three tissue samples obtainied from different mice, and comparable
results were obtained. The intensity of fluorescence is shown by colors
(the upper is the stronger). (Original magnification × 2,500.)
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| Fig 4.
Cellular response of +/+, mi/mi,
Mior/Mior and
Miwh/Miwh CMCs at various
concentrations of rmSCF was measured with means of MTT colorimetric
assay. Three independent experiments were performed with comparable
results, and the result of a representative experiment is shown. Each
point represents the mean of triplicate samples.
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The subcellular localization of MITF was examined by
immunocytochemistry. 293T cells that were transfected with expression vector containing +-MITF, mi-MITF, or-MITF, or
wh-MITF cDNA were stained with the anti-MITF polyclonal
antibody. Strong signals were detected in the nuclei of the cells
overexpressing +-MITF, or-MITF, or wh-MITF cDNA
(Fig 5). In contrast, signals were
predominantly detected in the cytoplasm of the cells overexpressing
mi-MITF cDNA (Fig 5).
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| Fig 5.
Subcellular localization of MITF determined by
immunocytochemistry. 293T cells were transfected with expression vector
containing +-MITF (A), mi-MITF (B), or-MITF (C), or
wh-MITF (D) cDNA. After 48 hours of transfection, cells were
stained with anti-MITF antibody. (Original magnification × 4,000.)
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The DNA binding ability of or-MITF and wh-MITF was
examined using EGMSA. The oligonucleotide containing the CACATG motif, which represents a part of MMCP-6 promoter, was used as the
probe.24 No DNA-protein complex was detected when
or-MITF or mi-MITF was incubated with the protein. The
specific binding of +-MITF and wh-MITF was detected, but the
amount of DNA-wh-MITF complex was smaller than the amount of
DNA-+-MITF complex (Fig 6A). When the dissociation assay was performed, the dissociation rate of
DNA-wh-MITF complex was higher than that of DNA-+-MITF complex
(Fig 6B). In other words, the affinity of wh-MITF to DNA was
lower than that of +-MITF.
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| Fig 6.
DNA binding ability of +-MITF, mi-MITF,
or-MITF, or wh-MITF examined by EGMSA and dissociation
assay performed with +-MITF or wh-MITF. (A) EGMSA using
GST-MITF fusion proteins. The labeled
5'-TGGTGGGGACACATGTTACATGGA oligonucleotide was used as
a probe (hexameric motif recognized by MITF is underlined). Each lane
contains 3.5 µg of GST-+-MITF, GST-mi-MITF,
GST-or-MITF, or GST-wh-MITF in the absence or presence
of a 200-fold molar excess of unlabeled oligonucleotide as a
competitor. Coomasie brilliant blue staining for GST-+-MITF,
GST-mi-MITF, GST-or-MITF, or GST-wh-MITF was
shown below the result of EGMSA. The amount of protein in each lane was
the same as the amount used in EGMSA. (B) Dissociation assay performed
with +-MITF and wh-MITF. Reaction mixtures for DNA-+-MITF
and DNA-wh-MITF complexes were prepared, and a 500-fold molar
excess of unlabeled oligonucleotide was added. The reactions were
sampled after further incubation time of 30, 60, 90, 120, and 150 seconds. The regions of the autoradiogram containing DNA-MITF complexes
were scanned with a densitometer, and the values were plotted against
the incubation period.
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Because the affinity of transcription factors to DNA might be affected
by the surrounding sequences of the core binding motif,45 there is a possibility that the affinity of each mutant MITF to the
CANNTG motif located in the MMCP-4, c-kit, Gr B, or TPH
promoter was different from that of the CANNTG motif located in the
MMCP-6 promoter. We examined the affinity of each mutant MITF to the CANNTG motif located in the MMCP-4, c-kit, Gr B, or TPH
promoter. We did not examine the affinity to the CAGTTG motif located
in the MMCP-5 promoter, because even +-MITF did not bind the
motif.19 The result observed in the MMCP-4, c-kit,
Gr B, or TPH promoter was comparable to the result observed in the
MMCP-6 promoter (Fig 7).
View larger version (70K):
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| Fig 7.
DNA binding ability of +-MITF, mi-MITF,
or-MITF, or wh-MITF to the binding motif located in
MMCP-6, MMCP-4, c-kit, Gr B, or TPH promoter. Each lane
contains 3.5 µg of GST-+-MITF, GST-mi-MITF,
GST-or-MITF, or GST-wh-MITF.
|
|
| |
DISCUSSION |
We compared the phenotype of mast cells among +/+, mi/mi,
Mior/Mior, and
Miwh/Miwh mice. As reported
previously,16-22 the phenotype of mast cells with
mi/mi genotype was abnormal in various items. In the skin of
mi/mi mice, mast cells decreased in number and the proportions of berberine sulfate-positive, MMCP-4 mRNA+, and MMCP-6
mRNA+ mast cells decreased remarkably. The expression of
c-kit protein also decreased in the skin mast cells of
mi/mi mice. Moreover, in mi/mi CMCs, the expression of
MMCP-4, MMCP-5, MMCP-6, c-kit, Gr B, or TPH gene was hardly
detectable, and the surface expression of c-kit receptor and
the response to rmSCF were severely deficient. The serotonin content
decreased significantly in mi/mi CMCs. The phenotype of mast
cells of Mior/Mior genotype was
abnormal in the same items as detected in mast cells of mi/mi
genotype. However, the magnitude of abnormality of
Mior/Mior mast cells was significantly
milder than that of mi/mi mast cells in the following items:
the number of skin mast cells in day-18 pc embryos, the proportion of
berberine sulfate-positive skin mast cells, the expression of
c-kit and Gr B mRNAs, and the surface expression of
c-kit protein in CMCs. The phenotypes of
Miwh/Miwh skin mast cells and CMCs were
mostly comparable to those of +/+ skin mast cells and CMCs, but the
decreased expression of Gr B and TPH mRNAs and the decreased serotonin
content were observed in Miwh/Miwh
CMCs. The abnormality of mast cells was severe in mi/mi mice, moderate in Mior/Mior mice, and slight
in Miwh/Miwh mice. The degree of
microphthalmia was severe in mi/mi and
Mior/Mior mice and moderate in
Miwh/Miwh mice.4
Osteopetrosis was severe in mi/mi mice, mild in
Mior/Mior mice, and undetectable in
Miwh/Miwh mice.4,26 The
rank order of abnormality in mast cells (mi/mi > Mior/Mior > Miwh/Miwh) was the same as that of the
severity observed in microphthalmia and osteopetrosis.
The expression level of c-kit protein in
Miwh/Miwh CMCs was comparable to that
of +/+ CMCs, but that of Mior/Mior and
mi/mi CMCs was significantly decreased. This was consistent with the cellular response to SCF, the ligand for c-kit
receptor tyrosine kinase. The rmSCF induced a dose-dependent response
in +/+ and Miwh/Miwh CMCs but not in
Mior/Mior and mi/mi CMCs.
The number of mast cells in the skin appeared to parallel with the
expression level of c-kit. The number of skin mast cells in
day-18 pc mi/mi embryos was smaller than the number in day-18 pc Mior/Mior embryos, and the
c-kit expression level of mast cells was also lower in day-18
pc mi/mi embryos than in day-18 pc
Mior/Mior embryos. The number of skin
mast cells and the expression level of c-kit was comparable
between 20-day-old mi/mi and
Mior/Mior mice. This is consistent with
our previous result observed in Wsh/Wsh
mice.46 Both the number of skin mast cells and the
expression level of c-kit decreased in an age-dependent manner
in Wsh/Wsh mice.
The functional level of MITF necessary for the mRNA expression of each
gene may be estimated by comparing +/+, mi/mi,
Mior/Mior, or
Miwh/Miwh mast cells. The
or-MITF was deficient in DNA binding ability but normal in
nuclear localization potential. The wh-MITF had normal nuclear
localization potential. The wh-MITF can bind DNA, but its
affinity to DNA was lower than that of +-MITF.
Miwh/Miwh CMCs normally expressed
MMCP-4, MMCP-6, and c-kit genes but weakly expressed TPH and Gr
B genes. The affinity of wh-MITF to the promoter of each gene
may be affected by the surrounding sequences, as reported by Fisher et
al45 in the case of the interaction between the Myc protein
and its targets. However, no significant difference was detected among
the affinity of wh-MITF to the CANNTG motif of MMCP-4, MMCP-6,
c-kit, Gr B, and TPH promoters. Therefore, the deficient
expression of Gr B and TPH genes may not be explained by the reduced
affinity of wh-MITF to the CANNTG motifs. The relatively weak
affinity of wh-MITF to the CANNTG motif may be enough for the
normal expression of MMCP-4, MMCP-6, and c-kit genes but not for TPH and Gr B genes.
The DNA binding potential of wh-MITF was deficient, but it was
apparently demonstratable. This was inconsistent with the finding of
Hemesath et al5 that wh-MITF did not bind to the
adenovirus major late promoter having the CACGTG motif as a core
sequence. We produced wh-MITF as a GST fusion protein in
Escherichia coli, whereas Hemesath et al5 produced
wh-MITF using reticulocyte lysate. We suppose that the purity
of wh-MITF used in our experiment was higher than that of
Hemesath et al,5 because we purified the
GST-wh-MITF fusion protein by affinity chromatography on
immobilized glutathione. There is a possibility that the relatively
weak affinity of wh-MITF to DNA was not detected by Hemesath et
al5 due to the lower purity of the wh-MITF used in
their experiment. There is another possibility that the difference
between the result of Hemesath et al5 and our result may be
attributable to the difference of probes.
Taken together, mast cells are suitable model for evaluating missing
functions of MITF encoded by various mutant alleles.
| |
ACKNOWLEDGMENT |
The authors thank Dr S. Nomura (Osaka University) for valuable
discussions, Dr Lynn Lamoreux for
Mior/Mior mice, Dr S. Nagata for
pEF-BOS, and Kirin Brewery Co Ltd for rmSCF.
| |
FOOTNOTES |
Submitted October 27, 1998; accepted February 17, 1999.
Supported by grants from the Ministry of Education, Science and
Culture, the Ministry of Health and Welfare, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, the Joint Research
Project under the Japan-Korea Basic Scientific Promotion Program, and
the Organization for Pharmaceutical Safety and Research.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Yukihiko Kitamura, MD, Department of
Pathology, Osaka University Medical School, Yamada-oka 2-2, Suita
565-0871, Osaka, Japan.
| |
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ABSTRACT
INTRODUCTION